This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The COBRE Imaging and Physiology Core maintains a wide range of microscopy and imaging systems configured especially for live-cell immunofluorescence preparations, with the capacity for routine fixed samples imaging as well. Our systems include a Noran/Prairie Technologies fast-scanning LSCM (laser scanning confocal microscope), a DeltaVision RT restoration microscopy system, a Zeiss/BioRad Radiance 2100 dedicated multiphoton LSCM, a Nikon TIRF (total-internal reflection fluorescence) system, a Lambda DG-4 ratiometric imaging system, an Andor iXon/Olympus widefield fluorescence microscope, and a Nikon SMZ-1500 fluorescence stereoscope. Patch-clamp electronics are available for use on most microscope systems, and each system has an integrated perfusion setup. The Noran is capable of high-speed image acquisition and is used primarily for calcium imaging on whole tissues and isolated cells. The DeltaVision system has a temperature and CO2-controlled incubator allowing for time-lapse experiments on cultured cells, and also excels at immunofluorescence and colocalization experiments on fixed samples. The Radiance 2100 Multiphoton features a Coherent Chameleon femtosecond pulsed IR laser and a fixed-stage upright Olympus microscope. The pulsed infrared excitation, coupled with non-descanned detectors, make it possible to image at depths of up to 500 microns for several hours. It is currently being used for calcium-imaging and uncaging experiments in brain-slices as well as retrograde labeling of neurons in the spinal cord of developing chick embryos. The ratiometric imaging system is used for imaging of fura-2 loaded cells or cells transfected with the FRET-based calcium indicator Cameleon. The TIRF system is used mainly for calcium imaging near the cell membrane on patch-clamped rodent cells. The Andor iXon system is a high-speed EM CCD coupled with an Olympus microscope for fluorescent image capture, and is currently used to image rhythmic activity in smooth muscle tissue preparations. For data deconvolution and analysis, we have two dedicated server workstations running softWoRx (Applied Precision) for deconvolution, MetaMorph (Universal Imaging), Volocity (Perkin Elmer) and ImageJ (NIH) for general analysis and rendering. The EM CCD workstation includes Andor iQ-CORE analysis software. Equipment scheduling is accessed online, and user hours are recorded in the core database.